Method of producing modified virus or vaccine



Patented Apr. 20, 1954 METHOD OF PRODUCING MODIFIED VIRUS B VACCINEArthur L. Brueckner, Hyattsville, and Reginald L. Reagan, Silver Spring,Md., assignors-to The University of Maryland, College Park, Md.

No Drawing. Application January 17, 1952, Serial No. 267,002

3 Claims. (Cl. 167-78) This application is a continuation-in-part of ourcopending application Serial Number 791,148, filed. December 11, 1947,now abandoned.

This invention relates to a modified virus or vaccine for use inimmunizing fowls, particularly chickens, against Newcastle disease. Thisdisease, also known as pneumoencephalitis in the United States and inother English speaking countries and under various other names in otherparts of the world, has been introduced into the United States in recentyears. Extensive studies where the disease is known prove that thecausative agent is a filter passing virus. The main symptoms are thosearising from the effects of the virus upon the respiratory and centralnervous systems. There are generally diflicult breathing, with wheezing,sneezing and coughing, and discharges from nostrils and mouth; loss ofappetite; dullness, weakness of one or both legs followed by paralysis;drooping of wings followed by paralysis; abnormal positions of the headand neck; and complete prostration. ihe death rate observed in flocks inthe United States has been variable, ranging up to 50% or more. There isprobability of greater death rate, since, in other countries, themortality has reached 100%.

Some work has been done heretofore in an an effort to develop a vaccinefor effectively immunizing chickens against Newcastle disease. Work inthe Philippines with a killed or inactivated vaccine has been describedby Aceveda and Mendoza. Others who have described investigations of theeffectiveness of killed vaccine in connection with the treatment orprevention of Newcastle disease were Nakamura and Wagtsuma in Japan;and. Beach in California. Haddow and Idnani described tests made with atemperature-attenuated vaccine for vaccination against Ranikhet disease,the name 'by which Newcastle disease is known in India. Komarov andGoldsmit (Palestine) published a preliminary observation on themodification of a strain of Newcastle disease virus by intracerebralpassage through ducklings. Generoso and Aceveda described thecultivation of avian pest virus in the embryos of chickens. So far as weknow, none of the vaccines heretofore provided have been effective tothe point of immunizing a large percentage of vaccinated chickensagainst Newcastle disease.

An object of the present invention is to provide an improved vaccine forimmunizing chickens against Newcastle disease.

Another object of the invention is to provide an improved method ofproducing such a vaccine.

Another object of the invention is to prepare from the virus ofNewcastle disease, a modified virus, or vaccine, capable of immunizingchickens against the disease, and which modified virus or vaccine isnon-pathogenic or has a. low degree of pathogenicity.

We have discovered that by passing a virus of Newcastle disease seriallythrough hamsters (small mammals native to Syria and not commonly knownin America), the virus may be so modified that it may be used safely,while still alive, for eiiectively immunizing chickens against Newcastledisease. The virus, now known as California strain 11914, used wasobtained from the Bureau of Animal Industry, United States Department ofAgriculture, Washington, D. C.

The serial transmission through hamsters was accomplished by inoculatinghamsters intracerebrally with the live aforementioned virus, thenpreparing a suspension of the ground brains of hamsters which died orbecome ill as a result of the injection, and then injecting other hamsters intracerebrally with the suspension. This procedure was carried onserially for a plurality of passages in accordance with the well knownor standard methods of modifying a virus by adaptation to a host. Testswere made by vaccinating healthy chickens with the hamsteradaptedvaccine and then challenging unvaccinated control chickens and chickensvaccinated with vaccine modified by different numbers of passagesthrough hamsters. It was found that the virus, thus modified byseventeen passages through hamsters, is useful for providing effectiveimmunization, but is sufficiently pathogenic to not be most advantageousfor all uses. Highly effective immunization with lower pathogenicity wasobtained with the hamster adapted Newcastle virus of the twenty-ninthand the fortyninth passages, the forty-ninth passage product having thelowest pathogenicity.

In general, the preparation of the modified Newcastle disease virusvaccine in accordance with our invention is somewhat analogous to knownprocedures, for example, the preparation of smallpox vaccine by themodification of human smallpox virus by serial passage through rabbitsor calves. However, it is not possible to predict that a particularvirus can be modified in this manner; and even if the adaptability toserial passage modification is assumed, it is not possible toforeseewhat animal or fowlcould serve as the host.

In the preparation of our improved vaccine, each of several hamsters wasinjected intracerebrally with one-tenth of 1 cc. of allantoic fluidinfected with the live Newcastle disease virus referred to. Symptoms ofirritability and malaise appeared three to six days after inoculation.During the moribund period, the animals showed labored breathing andmoved only slightly when disturbed. The hamsters from which brainmaterial was used for passage were sacrificed while moribund; and a tenpercent brain suspension was prepared by grinding the brain tissue withalundum and diluting with physiologic saline. Thereafter, eighthundredths to onetenth cc. of brain suspension was passed seriallythrough hamsters.

One-half cubic centimeter of a 1-0 per-cent infected brain suspension ofthe seventeenth passage of the aforementioned virus was injected intothe base of the wattle of each of a group of six-week-old chickens. Ninedays after inoculation, one-fourth of the injected chickens showedtypical Newcastle disease symptoms. The remaining chickens appearednormal for thirty days after inoculation. On the thirtieth day afterinoculation, they were challenged with 0.2 cc. of a dilution of avirulent egg-adapted strain of the virus, which titred 10- in ten-dayembryonated eggs. The virus also titered 10- in young chickens six weeksold. Another group of chickens of the same age as those injected withthe hamster-adapted Newcastle virus were used as controls. Both groups,the vaccinated group and the control group were challenged with the sameamount, that is, 0.2 cc. of 10- dilution of the egg-adapted virus. Nocharacteristic symptoms of Newcastle disease appeared in the vaccinatedgroup within an observation period of a month after the day ofchallenge. A11 of the chickens of the control group died of Newcastledisease symptoms on the fourth and sixth days after challenge. Thevaccine produced from the hamster-adapted virus of the seventeenthpassage, although having a substantial pathogenicity, can be used toadvantage in protecting major portions of flocks endangered by anunusually severe epidemic, since without such protection substantiallyentire flocks could succumb to the disease.

Further investigations with chickens inoculated with the hamster-adaptedvirus of the twenty-ninth and the forty-ninth passages were conducted.One hundred fifty healthy, twelve week old chickens, which had nohistory and had showed no indication of infection with Newcastledisease, were used for the first two large scale tests. Forty-two ofthese chickens, to be referred to as Group I. were injected at the baseof the wattle with .5 cc. of a 10 percent suspension of infected hamsterbrain of the twenty-ninth passage. Eight uninjected chickens were keptwith this group as room controls. About one week after injection, fourof the inoculated chickens showed symptoms typical of Newcastle disease.Two died within forty-eight hours of injectionv and were consideredatypical. The pathogenicity of the twenty-ninth passage virus was thusabout 9 percent, on the basis of typical symptoms. All of the roomcontrols survived and were still healthy twenty-eight days afterinjection.

On the twenty-eighth day after injection, the room controls and thethirty-six surviving test birds were challenged with .2 cc. of virulentchallenge virus injected into the base of the wattle.

Concentrations of the challenge virus for each 0! three sub-groups oftwelve surviving chickens of Group I were 10- 10- and 10*. Challengevirus titration established a concentration of 10- as that which wouldcause death of fifty percent of susceptible chickens. Following thechallenge, two of the birds previously vaccinated with thehamster-adapted Newcastle virus, twenty-ninth passage, died. Thirty-four(94.5 percent) of the vaccinated birds survived.

A similar test was made with a second group of fifty chickens, forty-twobeing vaccinated with twenty-ninth passage hamster-adapted Newcastlevirus, and the other eight being kept with the vaccinated birds as roomcontrols. The times of administering, and the size and concentration ofthe dosages were the same as in the Group I tests, but the vaccinationswere made in the breast muscles instead of the Wattles. The challengewas the same as the Group I challenge. The Group II results were thesame as the Group I results with the exception that four, instead oftwo, of the vaccinated birds succumbed to the challenge.

Thirty chickens of the original flock of one hundred fifty were used asnon-contact controls, 1. e., were kept separate from the chickens ofGroups I and II, but were challenged in the same manner as the Groups Iand II vaccinated and room control chickens. Of the thirty non-contactcontrols, only three, or 10 percent survived the challenge. The roomcontrol birds kept in contact with the Groups I and II vaccinated birdsshowed no evidence of Newcastle disease during the twenty-eight dayperiod prior to challenging the test birds. This shows clearly that themodified virus or vaccine does not create, in vaccinated birds, adisease transmissable clinically to non-vaccinated healthy birds bycontact with vaccinated birds. On the contrary, contact of the healthyunvaccinated room control birds with the vaccinated birds apparentlyproduced a measurable resistance to the challenge, as shown by the factthat 62.5% of the challenged room control birds survived, whereas only10 percent of the normal, healthy, unvaccinated birds used asnon-contact challenge controls survived.

The remaining twenty of the flock of one hundred fifty chickens used inthe tests referred to above were employed for titration of challengevirus. Still better results, particularly as to lower pathogenicity, areobtainable with a vaccine comprising hamster-adapted Newcastle diseasevirus of passages beyond the twenty-ninth passage. In a representativefurther test in which hamster-adapted Newcastle disease virus of theforty-ninth passage was used, thirty chickens were injected in thewattle with .5 cc. of the vaccine, thirty-seven chickens were similarlyinjected with .25 cc., and thirty-seven chickens were injected similarlywith .1 cc. Or this entire, Group III of one hundred four vaccinatedtest birds, 1.9 percent developed typical Newcastle disease symptomsprior to being challenged,

About percent of the challenged vaccinated Only twenty-f controls and.

birds survived a heavy challenge. five percent of the challenged room 3percent of the non-contact controls challenged with the same dose as theGroup III vaccinated. The 001106111.

test birds survived the challenge. trations of the challenge doses werethe same as used in the Groups I and II tests, but the size of the dosewas .25 cc., somewhat larger than the. .2 cc. challenge dose used in theGroups I and II tests.

Vaccine produced from the allantoic fluid of chick embryos injected withforty-ninth passage hamster-adapted Newcastle virus was injected intoninety-three chickens of Group IV. These chickens were obtained from asource other than the source of the Groups I, II, and III test chickensand the chickens of a fifth group to be referred to later. It wasobserved, before making the tests, that the Group IV test chickens werenot as healthy as the other test chickens. Ninety-two per cent of thechallenged vaccinated test chickens of Group IV survived the challenge;but the pathogenicity of the Group IV vaccine was somewhat higher thanthat of the hamsteradapted virus vaccine used in the testing of thebirds of Groups I, II and III. Only 40 per cent of the challenged roomcontrols and 3 per cent of the non-contact controls challenged with thesame dose as the Group IV vaccinated birds survived the challenge. Thevaccine used in the *roup IV tests was prepared by inoculating elevenday old embryonated eggs with .1 cc. of re per cent hamster brainsuspension, incubating the eggs at 37 C. for three days, and pooling theallantoic fluid from embryonated eggs which appeared sluggish. Thisfluid titered 10- in eleven day embryonated eggs. A 10 per centsuspension of the allantoic fluid was used for vaccinating the Group IVtest birds in the Wattles, fifty-one birds being vaccinated with .5 cc.,and forty-two birds with .25 cc. of the vaccine.

Another vaccine was produced from a sheep brain suspension prepared byinjecting a ewe intracerebrally with 5 cc. of 10 per cent hamsteradaptedNewcastle virus oi the thirty-sixth passage. On the eighth day afterinoculation the sheep became moribund. Shortly after its death the brainwas removed and 10 per cent suspensicn was prepared with physiologicalsaline. This suspension, which titrated 10- in hamsters, was used forvaccination of ninety chickens of Group V, forty chickens being injectedin the wattle with .5 cc., and fifty in the wattle with .25 cc. Thepathogenicity of the sheep brain suspension vaccine was nil, but theimmunizing properties were not as potent as those of the vaccines usedin the tests of the Groups I, II, III, and IV chickens. Nevertheless,considerably more than 50 per cent of the Group V vaccinated test birdssurvived the heavy challenge, whereas only 25 per cent of the challengedroom controls and about 3 per cent of the challenged non-contact controlbirds survived the challenge. The challenge was the same as the Group IVchallenge.

Hamster-adapted Newcastle disease virus vaccine is efiective forimmunization against contraction of the disease from contact withinfected chickens as well as for protecting or immunizing against achallenge dose administered by injection. Twenty-eight days aftervaccination of the test chickens of Groups III and V, and before any ofthese chickens were challenged by injection, twenty-three of thevaccinated chickens were placed with twenty-two normal, non-infected andunvaccinated chickens, and twenty unvaccinated chickens which had beeninfected with Newcastle disease. The twenty infected chickens were beingused at this time for challenge virus titration of 10- and 10-dilutions. The twenty-three vaccinated chickens included five from GroupIII vaccinated with .5 cc. of hamster-adapted virus of the fortyninthpassage, five from Group III vaccinated with .25 cc. of the samevaccine, five from Group V vaccinated with .5 cc. of the Newcastlemodified virus sheep brain suspension, and eight vaccinated with .25 cc.of the sheep brain suspension vaccine. None of the vaccinated chickensshowed symptoms of Newcastle disease, whereas within twenty-one daysafter exposure to the twenty infected chickens, nine of the twentytwounvaccinated and originally uninfected chickens showed typical Newcastledisease symptoms.

The unvaccinated room control chickens were exposed to the vaccinatedchickens or all of Groups I, II, III, IV, and V until the day ofchallenge, but none of these control birds developed Newcastle diseaseprior to the challenge injection.

Newcastle disease virus modified by serial passage through hamsters inaccordance with the present invention may be used in accordance standardmethods for the commercial manufacture of vaccines. The specificprocedures described above are intended to be under stood asrepresentative of, rather than definitive of the invention.

We claim:

1. In a method of modifying California strain 11914 Newcastle diseasevirus for the production of a vaccine capable when injected intochickens. of immunizing them against the disease; inoculating a hamsterwith said virus; obtaining the virus in modified state from the brain ofsaid hamster; and serially passing the modified virus through the brainsof other hamsters for not less than twenty-nine passages.

2. In a method of modifying California strain 11914 Newcastle diseasevirus for the production or" a vaccine capable when injected intochickens, of immunizing them against the disease; inoculating a hamsterwith said virus; obtaining the virus in modified state from the brain ofsaid hamster; and serially passing the modified virus through the brainsof other hamsters for not less than forty-nine passages.

3. In a method of modifying California strain 11914 Newcastle diseasevirus for the production of a vaccine capable when injected intochickens, of immunizing them against the disease; inoculating a hamsterwith said virus; obtaining the virus in modified state from the brain ofsaid hamster; and serially passing the modified virus through the brainsof other hamsters for not less than seventeen passages.

References Cited in the file of this patent UNITED STATES PATENTS NumberName Date 2,136,131 Green Nov. 8, 1938 2,271,819 Green Feb. 3, 1942OTHER REFERENCES Beveridge: The Cultivation of Viruses and Rickettsiaein the Chick Embryo, London, 1946, page 58.

Brandley et al.: Am. J. Vet. Res., vol. 7, pp. 298, 304, 305, 307 to322, July 1946.

Zinssers Textbook of Bacteriology (1948), page 806.

3. IN A METHOD OF MODIFYING CALIFORNIA STRAIN 11914 NEWCASTLE DISEASEVIRUS FOR THE PRODUCTION OF A VACCINE CAPABLE WHEN INJECTED INTOCHICKENS, OF IMMUNIZING THEM AGAINST THE DISEASE; INOCULATING A HAMSTERWITH SAID VIRUS; OBTAINING THE VIRUS IN MODIFIED STATE FROM THE BRAIN OFSAID HAMSTER; AND SERIALLY PASSING THE MODIFIED VIRUS THROUGH THE BRAINSOF OTHER HAMSTERS FOR NOT LESS THAN SEVENTEEN PASSAGES.